The present invention relates to anti-inflammatory compounds that act via inhibition of Monocyte Chemoattractant Protein-1 (MCP-1) and especially MCP-1 inhibitor compounds that contain an indole moiety. The invention further relates to processes for their preparation, to intermediates useful in their preparation, to their use as therapeutic agents and to pharmaceutical compositions containing them.
MCP-1 is a member of the chemokine family of pro-inflammatory cytokines which mediate leukocyte chemotaxis and activation. MCP-1 is a C-C chemokine which is one of the most potent and selective T-cell and monocyte chemoattractant and activating agents known. MCP-1 has been implicated in the pathophysiology of a large number of inflammatory diseases including rheumatoid arthritis, glomerular nephritides, lung fibrosis, restenosis (International Patent Application WO 94/09128), alveolitis (Jones et al., 1992, J. Immunol., 149, 2147) and asthma. Other disease areas where MCP-1 is thought to play a part in their pathology are atherosclerosis (e.g. Koch et al., 1992, J. Clin. Invest., 90, 772-779), psoriasis (Deleuran et al., 1996, J. Dermatological Science, 13, 228-236), delayed-type hypersensitivity reactions of the skin, inflammatory bowel disease (Grimm et al., 1996, J. Leukocyte Biol., 59, 804-812), multiple sclerosis and brain trauma (Berman et al, 1996, J. Immunol., 156, 3017-3023). An MCP-1 inhibitor may also be useful to treat stroke, reperfusion injury, ischemia, myocardial infarction and transplant rejection.
MCP-1 acts through the MCP-1 receptor (also known as the CCR2 receptor). MCP-2 and MCP-3 may also act, at least in part, through the MCP-1 receptor. Therefore in this specification, when reference is made to xe2x80x9cinhibition or antagonism of MCP-1xe2x80x9d or xe2x80x9cMCP-1 mediated effectsxe2x80x9d this includes inhibition or antagonism of MCP-2 and/or MCP-3 mediated effects when MCP-2 and/or MCP-3 are acting through the MCP-1 receptor.
Patent Nos. U.S. 005389650A, U.S. 005290798A, EP 0535926A1, EP 0535923A1, U.S. 005190968A, EP 0535924A1, EP 0419049A1, U.S. Pat. No. 5,308,850, EP 0535925A1, WO 93/16069, WO 93/25546, U.S. 005273980A and U.S. Pat. No. 5,272,145 disclose indole compounds as inhibitors of leukotriene biosynthesis with a benzyl moiety attached to the nitrogen of the indole ring. Similar compounds are also disclosed in WO 93/20078 (severe head injury), EP 0186367 (antiallergy), EP 0275667 (inhibitors of leukotriene biosynthesis), U.S. 4965369A (process patent) WO 94/14434 (antagonizing endothelin receptors), EP 0480659 A2 (treatment of hyperuricemia) and WO 96/03377A1 (allosteric effectors at muscarinic receptors).
The present invention is based on the discovery of a class of compounds containing an indole moiety which have useful inhibitory activity against MCP-1.
Accordingly one aspect of the present invention provides the use of a compound of the formula (I) in the manufacture of a medicament for antagonising an MCP-1 mediated effect in a warm blooded animal, such as man; 
wherein:
R1 is independently selected from trifluoromethyl, C1-4alkyl, halo, hydroxy, C1-4alkoxy, C1-4alkanoyl, C1-4alkanoyloxy, carboxy, trifluoromethoxy, amino, cyano, C1-4alkylamino, di(C1-4alkyl)amino, C1-4alkanoylamino, nitro, carbamoyl, C1-4alkoxycarbonyl, thiol, C1-4alkylsulphanyl, C1-4alkylsulphinyl, C1-4alkylsulphonyl, sulphonamido, carbamoylC1-4alkyl, Nxe2x80x94(C1-4alkyl)carbamoylC1-4alkyl, Nxe2x80x94(C1-4alkyl)2carbamoyl-C1-4alkyl, hydroxyC1-4alkyl, C1-4alkoxyC1-4alkyl, morpholino, pyrrolidinyl, carboxyC1-4alkylamino, R3 and xe2x80x94OR3 (where R3 is optionally substituted aryl or an optionally substituted 5- or 6- membered heteroaryl ring);
p is 0-4 and R1 can have the same or different values when p is 2-4 with the proviso that no more than one R1 can be chosen from the group amino, C1-4alkylamino, di(C1-4alkyl)amino, morpholino and pyrrolidinyl;
T is of the formula
xe2x80x94(CHR4)mxe2x80x94,
where
R4 is independently selected from hydrogen or C1-4alkyl and m=1-3 and R4 can have different values when m is 2 or 3;
X is CO2R4, SO3H, cyano, xe2x80x94SO2NHR4 (where R4 is as defined above), xe2x80x94SO2NHAr (where Ar is an optionally substituted phenyl or optionally substituted 5 or 6 membered heteroaryl ring), xe2x80x94CONHR5 (where R5 is H, cyano, C1-4alkyl, OH, xe2x80x94SO2xe2x80x94C1-4alkyl, xe2x80x94SO2CF3, xe2x80x94SO2-phenyl, xe2x80x94(CHR4)rxe2x80x94COOH, (where r is 1-3 and R4 (as defined above) can take different values when r is 2-3)), or X is a group of formula (II) 
or X represents a group of formula (III) 
where the groups defined as R4 may have different values within the definition of R4 above;
A is selected from phenyl, naphthyl, furyl, pyridyl and thienyl;
R2 is independently selected from trifluoromethyl, C1-4alkyl, halo, hydroxy, CF3Oxe2x80x94, C1-4alkoxy, C1-4alkanoyl, C1-4alkanoyloxy, amino, cyano, C1-4alkylamino, (C1-4alkyl)2amino, C1-4alkanoylamino, nitro, carboxy, carbamoyl, C1-4alkoxycarbonyl, thiol, C1-4alkylsulphanyl, C1-4alkylsulphinyl, C1-4alkylsulphonyl, sulphonamido, carbamoylC1-4alkyl, Nxe2x80x94(C1-4alkyl)carbamoylC1-4alkyl, Nxe2x80x94(C1-4alkyl)2carbamoyl-C1-4alkyl, hydroxyC1-4alkyl, C1-4alkoxyC1-4alkyl or two R2 values together may form a divalent radical of the formula xe2x80x94O(CH2)1-4Oxe2x80x94 attached to adjacent carbon atoms on ring A;
q is 0-4 and R2 can have the same or different values when q is 2-4;
Z is hydrogen, fluoro, chloro, bromo, iodo, methyl, trifluoromethyl, hydroxymethyl, acetyl, carboxyC3-6cycloalkyl or xe2x80x94(CHR4)rxe2x80x94NR6R7 (where r is 0-2, R6 and R7 are independently selected from H and C1-4alkyl or R6 and R7 together with the nitrogen to which they are attached form a 5 or 6 membered non-aromatic ring optionally containing one further heteroatom selected from O, N or S);
or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof.
A further aspect of the present invention provides the use of a compound of the formula (I) in the manufacture of a medicament for antagonising an MCP-1 mediated effect in a warm blooded animal, such as man wherein R1 is independently selected from trifluoromethyl, C1-4alkyl, halo, hydroxy, C1-4alkoxy, C1-4alkanoyl, C1-4alkanoyloxy, amino, cyano, C1-4alkylamino, di(C1-4alkyl)amino, C1-4alkanoylamino, nitro, carbamoyl, C1-4alkoxycarbonyl, thiol, C1-4alkylsulphanyl, C1-4alkylsulphinyl, C1-4alkylsulphonyl, sulphonamido, carbamoylC1-4alkyl, Nxe2x80x94(C1-4alkyl)carbamoylC1-4alkyl, Nxe2x80x94(C1-4alkyl)2carbamoyl-C1-4alkyl, hydroxyC1-4alkyl, C1-4alkoxyC1-4alkyl, morpholino, pyrrolidinyl, carboxyC1-4alkylamino, R3 and xe2x80x94OR3 (where R3 is optionally substituted phenyl or an optionally substituted 5- or 6- membered heteroaryl ring);
p is 0-4 and R1 can have the same or different values when p is 2-4 with the proviso that no more than one R1 can be chosen from the group amino, C1-4alkylamino, di(C1-4alkyl)amino, morpholino and pyrrolidinyl; Z is hydrogen, fluoro, chloro, bromo, iodo, methyl, trifluoromethyl, hydroxymethyl, carboxyC3-6cycloalkyl or xe2x80x94(CHR4)rxe2x80x94NR6R7 (where r is 0-2, R6 and R7 are independently selected from H and C1-4alkyl or R6 and R7 together with the nitrogen to which they are attached form a 5 or 6 membered non-aromatic ring optionally containing one further heteroatom selected from O, N or S); and X, T, A, R2 and q have any of the values defined above; or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof.
In this specification the term xe2x80x98alkylxe2x80x99 includes straight chained, branched structures and ring systems. For example, xe2x80x9cC1-4alkylxe2x80x9d includes propyl, isopropyl, i-butyl and cyclopropane. However, references to individual alkyl groups such as xe2x80x98propylxe2x80x99 are specific for the straight chained version only, references to individual branched chain alkyl groups such as xe2x80x98isopropylxe2x80x99 are specific for the branched chain version only and references to the cyclo groups such as cyclopropane are specific to the cyclic groups only. A similar convention applies to other radicals, for example xe2x80x9chydroxyC1-4alkylxe2x80x9d includes 1-hydroxyethyl and 2-hydroxyethyl. The term xe2x80x9chaloxe2x80x9d refers to fluoro, chloro, bromo and iodo.
Suitable optional substituents for aryl and heteroaryl are any of the values defined for R1 and R2 above. xe2x80x9cArylxe2x80x9d means phenyl or naphthyl, preferably phenyl. xe2x80x9cHeteroarylxe2x80x9d means an aromatic mono- or bicyclic- 5-10 membered ring with up to three or five ring heteroatoms (in mono and bicyclic rings respectively) selected from nitrogen, oxygen and sulphur. Examples of xe2x80x9cheteroarylxe2x80x9d include thienyl, pyrrolyl, furanyl, imidazolyl, thiazolyl, pyrimidinyl, pyridinyl, indolyl, benzimidazolyl, benzthiazolyl, quinolyl and isoquinolinyl.
An example of xe2x80x9cC1-4alkanoyloxyxe2x80x9d is acetoxy. Examples of xe2x80x9cC1-4alkoxycarbonylxe2x80x9d include methoxycarbonyl, ethoxycarbonyl, n- and t-butoxycarbonyl. Examples of xe2x80x9cC1-4alkoxyxe2x80x9d include methoxy, ethoxy and propoxy. Examples of xe2x80x9cC1-4alkanoylaminoxe2x80x9d include formamido, acetamido and propionylamino. Examples of xe2x80x9cC1-4alkylsulphanylxe2x80x9d include methylthio and ethylthio. Examples of xe2x80x9cC1-4alkylsulphinylxe2x80x9d include methylsulphinyl and ethylsulphinyl. Examples of xe2x80x9cC1-4alkylsulphonylxe2x80x9d include methylsulphonyl and ethylsulphonyl. Examples of xe2x80x9cC1-4alkanoylxe2x80x9d include propanoyl and ethanoyl. Examples of xe2x80x9cC1-4alkylaminoxe2x80x9d include methylamino and ethylamino. Examples of xe2x80x9cdi(C1-4alkyl)aminoxe2x80x9d include di-N-methylamino, di-(N-ethyl)amino and N-ethyl-N-methylamino. Examples of xe2x80x9cC1-4alkoxyC1-4alkylxe2x80x9d methoxymethyl and propoxyethyl. Examples of xe2x80x9ccarbamoylC1-4alkylxe2x80x9d are methylcarboxamide and ethylcarboxamide. Examples of xe2x80x9ccarboxyC3-6cycloalkylxe2x80x9d are 2-carboxycyclopropyl and 3-carboxycyclopentyl. Examples of xe2x80x9cNxe2x80x94(C1-4alkyl)carbamoylC1-4alkylxe2x80x9d are methylaminocarbonylethyl and ethylaminocarbonylpropyl. Examples of xe2x80x9cNxe2x80x94(C1-4alkyl)2carbamoyl-C1-4alkylxe2x80x9d are dimethylaminocarbonylethyl and methylethylaminocarbonylpropyl. Examples of xe2x80x9ccarboxyC1-4alkylaminoxe2x80x9d are carboxy methyl and carboxypropyl.
A particular group of values for R1 includes, for example, trifluoromethyl, (1-4C)alkyl, halogeno, hydroxy, (1-4C)alkoxy, (1-4C)alkanoyl, carboxy, trifluoromethoxy, amino, (1-4C)alkanoylamino, nitro, (1-4C)alkylsulphonyl, carboxyC1-4alkylamino, acetyl, phenoxy, phenyl optionally bearing a dimethylamino, trifluoromethyl, fluoro, chloro, methoxy, methyl or amino group, naphthyl, thien-2-yl, 5-halogenothien-2-yl, thien-3-yl and pyridyl.
A particular value for p is 0, 1 or 2.
A particular group of values for Z includes, for example, hydrogen, fluoro, chloro, bromo, iodo, methyl, trifluoromethyl, hydroxymethyl, 2-carboxycyclopropyl, amino and acetyl.
A particular group of values for X includes, for example, carboxy, (1-4C)alkoxycarbonyl, cyano, xe2x80x94CONHR5 wherein R5 is xe2x80x94SO2CF3, SO2xe2x80x94C1-4alkyl or xe2x80x94SO2-phenyl.
A particular group of values for R2 includes, for example, trifluoromethyl, (1-4C)alkyl, halogeno, trifluoromethoxy, (1-4C)alkoxy and nitro.
Preferred values for R1, p, Z, X, T, A R2 and q are as follows.
Preferred values for R1 are C1-4alkoxy, halo, nitro, amino, phenoxy or trifluoromethyl, more preferably chloro and/or C1-4alkoxy. Another preferred value for R1 includes, for example, carboxymethylamino. Where R1 is halo, fluoro, chloro or bromo are preferred. Where R1 is C1-4alkoxy, methoxy or ethoxy are preferred, particularly methoxy. Preferably position 7 is unsubstituted, and preferably there is no more than one C1-4alkoxy group.
Preferably p is 1 or 2.
Preferred combinations of p and R1 are as follows.
When p=1 then R1 is preferably 4-methoxy, 4-phenyl, 4-amino, 5-chloro, 5-methoxy, 5-nitro, 5-bromo, 5-phenoxy, 5-fluoro, 5-amino, 6-fluoro, 6-trifluoromethyl, 6-nitro or 6-chloro more preferably 4-amino, 5-amino, 5-chloro or 6-chloro.
T is preferably xe2x80x94CH2xe2x80x94.
Preferably X is carboxy or xe2x80x94CONHR5 (where R5 is defined above). Preferably R5 is xe2x80x94SO2CF3. Most preferably X is carboxy.
Preferably A is phenyl, naphthyl, furyl and thienyl especially phenyl or thienyl. When A is thienyl it is preferably thien-2-yl. Most preferably A is phenyl.
R2 is preferably chloro, bromo, methyl, methoxy, nitro, trifluoromethyl or trifluoromethoxy. Another preferred value for R2 includes, for example, fluoro.
q is preferably 1 or 2, especially 2.
Preferred combinations of A, R2 and q are as follows.
When A is phenyl, and q is 1, then R2 is preferably chloro especially 3-chloro or 4-chloro. Another preferred value for R2 includes, for example, 3-fluoro, 4-fluoro or 3-trifluoromethyl.
When A is phenyl, and q is 2, then R2 is preferably chloro, especially 3,4-dichloro. Another preferred value for R2 includes, for example, fluoro, especially 3,4-difluoro.
When A is phenyl then the positions ortho to T are preferably unsubstituted.
When A is thien-2-yl then preferably R2 is chloro, especially 5-chloro.
Preferably Z is hydrogen, bromo or methyl, especially hydrogen.
A preferred class of compounds within formula (I) for use in the present invention is that of formula (Ixe2x80x2): 
wherein:
Re is methoxy, fluoro, chloro, bromo, nitro, amino, phenoxy, trifluoromethyl, carboxy or hydroxy;
x is 1 or 2 with the proviso that there is at most one methoxy group;
Xxe2x80x2 is carboxy, xe2x80x94CONHSO2CF3, xe2x80x94CONHEt or xe2x80x94CONHMe;
Axe2x80x2 is phenyl or thienyl;
Rf is chloro, bromo, methyl, methoxy, nitro, trifluoromethyl or trifluoromethoxy;
y is 1 or 2;
Zxe2x80x2 is hydrogen, methyl bromo or carboxycyclopropyl; or a pharmaceutically acceptable salt or in vivo hydrolysable ester thereof.
Re is particularly methoxy, fluoro, chloro, bromo, nitro, amino, phenoxy or trifluoromethyl.
Preferably Re is chloro, amino, fluoro, nitro or methoxy. Preferably position 7 is unsubstituted.
Preferred combinations of x and Re are as follows.
When x=1 then Re is preferably chloro, amino, fluoro, nitro or methoxy especially. 4-methoxy, 4-amino, 5-chloro, 5-methoxy, 5-nitro, 5-bromo, 5-fluoro, 5-amino, 6-fluoro, 6-nitro or 6-chloro and especially 4-methoxy or 4-amino.
When x=4 then Re is preferably F.
Zxe2x80x2 is preferably hydrogen.
Xxe2x80x2 is preferably carboxy.
Axe2x80x2 is preferably phenyl. Where Axe2x80x2 is thienyl it is preferably thien-2-yl.
Preferred combinations of Axe2x80x2, Rf and y are as follows.
When Axe2x80x2 is phenyl, and y is 1, then Rf is preferably chloro especially 3-chlorophenyl or 4-chlorophenyl.
When Axe2x80x2 is phenyl, and y is 2, then Rf is preferably chloro, especially 3,4-dichlorophenyl.
When Axe2x80x2 is phenyl then the positions ortho to the CH2 moiety linked to the indole ring are preferably hydrogen.
When Axe2x80x2 is thien-2-yl then preferably Rf is chloro especially 5-chloro.
A further aspect of the present invention is a novel compound of the formula (I) or (Ixe2x80x2) as defined above.
Accordingly a further aspect of the present invention provides a novel compound of the formula (A): 
which is an inhibitor of monocyte chemoattractant protein-1 and wherein:
Ra is 4-methoxy, 4-phenyl, 4-amino, 4-thien-2-yl, 5-chloro, 5-methoxy, 5-nitro, 5-bromo, 5-phenoxy, 5-fluoro, 5-carboxymethylamino, 5-amino, 6-fluoro, 6-trifluoromethyl, 6-nitro or 6-chloro;
c is 0, 1 or 2 provided that there is no more than one methoxy group;
W is hydrogen, bromo, methyl or transcyclopropyl-2-carboxylic acid;
G is phenyl or thien-2-yl;
When G is phenyl Rb is 3chloro, 3-trifluoromethyl, 3-nitro, 3-methoxy, 4-trifluoromethyl, 4-trifluoromethoxy or 4-chloro;
When G is thien-2-yl Rb is 5-chloro;
d is 1 or 2;
or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof.
Preferred values for Ra, c, G, Rb, W, A and d are as follows.
Preferred values for Ra are 4-methoxy, 4-amino, 5-fluoro, 5chloro, 6-chloro or 5-methoxy more preferably 4-amino or 5-fluoro.
Preferably c is 0 or 1 more preferably 1.
Preferred combinations of c and Ra are as follows.
When c=1 then Ra is preferably 4-amino, 4-thien-2-yl, 4-methoxy, 5-chloro, 5-fluoro, 5-amino, 6-fluoro or 6-chloro, but 4-amino, 4-thien-2-yl and 5-fluoro are most preferred.
W is preferably bromo or hydrogen, especially hydrogen.
Preferably G is phenyl.
Preferably d is 2.
When G is phenyl preferably Rb is 3-chloro, 3-trifluoromethyl or 4-chloro.
Preferred combinations of G, d and Rb are as follows.
When G is phenyl and d is 1, then G taken together with Rb is preferably 3-chlorophenyl, 4-chlorophenyl, 3-iodophenyl, 4-iodophenyl or 3-trifluoromethylphenyl, and especially 3-chlorophenyl, 4-chlorophenyl or 3-trifluoromethylphenyl.
When G is phenyl and d is 2 then G taken together with Rb is preferably 3,4-dichlorophenyl or 3,4-difluorophenyl, especially 3,4-dichlorophenyl.
A further aspect of the present invention provides a novel compound of the formula (B) 
wherein X2 is carboxy, xe2x80x94CONHSO2CH3 or xe2x80x94CONHSO2-phenyl; Z2 has any of the meanings, including particular and preferred values, for Z or Zxe2x80x2 defined herein; Rw and Rx are independently halogeno; Ry is independently selected from any of the meanings, including particular and preferred values, for R1 or Re defined herein; and t is 1 or 2; or a pharmaceutically acceptable salt thereof. Within this group of compounds, those in which Rw and Rx are both fluoro or both chloro are particularly preferred, and especially those in which Rw and Rx are both chloro. Compounds in which X2 is carboxy are also particularly preferred.
Compounds of formulas (I), (Ixe2x80x2), (A) and/or (B) which are of particular interest include, for example, the specific embodiments set out hereinafter in the accompanying examples, especially Examples 3 and 3.01 to 3.111 inclusive, and these compounds and/or the use of these compounds are provided as a further feature of the invention. Of these, preferred compounds include the compounds described in Examples 3, 3.01, 3.02, 3.03, 3.04, 3.08, 3.10, 3.11, 3.12, 3.13, 3.14, 3.16, 3.17, 3.18, 3.19, 3.20, 3.21, 3.22, 3.24, 3.25, 3.27, 3.28, 3.29, 3.30, 3.32, 3.33, 3.34, 3.35, 3.43, 3.44, 3.45, 3.46, 3.47, 3.48, 3.49, 3.50, 3.51, 3.52, 3.54, 3.55, 3.57, 3.58, 3.59, 3.60, 3.61, 3.62, 3.63, 3.64, 3.65, 3.66, 3.67, 3.68, 3.69, 3.70, 3.71, 3.72, 3.73, 3.75, 3.76, 3.77, 3.78, 3.79, 3.80, 3.81, 3.82, 3.83, 3.85, 3.86, 3.87, 3.88, 3.89, 3.90, 3.91, 3.92, 3.93, 3.94, 3.95, 3.97, 3.98, 3.99, 3.100, 3.101, 3.102, 3.103, 3.104, 3.105, 3.106, 3.107, 3.108, 3.109, 3.110 and 3.111, or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof. Of these, paticularly preferred compounds include the compounds described in Examples 3.02, 3.03, 3.11, 3.12, 3.14, 3.22, 3.30, 3.46, 3.54, 3.58, 3.59, 3.60, 3.61, 3.68, 3.69, 3.73, 3.82, 3.83, 3.86, 3.88, 3.90, 3.92, 3.93, 3.94, 3.100, 3.105, 3.106, 3.107, 3.108, 3.109 and 3.111, or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof.
Suitable pharmaceutically acceptable salts include acid addition salts such as methanesulfonate, fumarate, hydrochloride, hydrobromide, citrate, maleate and salts formed with phosphoric and sulphuric acid. In another aspect suitable salts are base salts such as an alkali metal salt for example sodium, an alkaline earth metal salt for example calcium or magnesium, an organic amine salt for example triethylamine, morpholine, N-methylpiperidine, N-ethylpiperidine, procaine, dibenzylamine, N,N-dibenzylethylamine or amino acids for example lysine. There may be more than one cation or anion depending on the number of charged functions and the valency of the cations or anions. A preferred pharmaceutically acceptable salt is a sodium salt.
Some compounds of formula (I) may possess chiral centres. It is to be understood that the invention encompasses all such optical isomers and diasteroisomers of compounds of formula (I).
The invention further relates to all tautomeric forms of the compounds of formula (A), (B) or formula (I).
It is also to be understood that certain compounds of formula (A), formula (B) or formula (I) can exist in solvated as well as unsolvated forms such as, for example, hydrated forms. It is to be understood that the invention encompasses all such solvated forms.
An in vivo hydrolysable ester of a compound of formula (A) or formula (I) containing carboxy or hydroxy group is, for example, a pharmaceutically acceptable ester which is hydrolysed in the human or animal body to produce the parent acid or alcohol.
Suitable pharmaceutically acceptable esters for carboxy include C1-6alkoxymethyl esters for example methoxymethyl, C1-6alkanoyloxymethyl esters for example pivaloyloxymethyl, phthalidyl esters, C3-8cycloalkoxy-carbonyloxyC1-6alkyl esters for example 1-cyclohexylcarbonyloxyethyl; 1,3-dioxolen-2-onylmethyl esters for example 5-methyl-1,3-dioxolen-2-onylmethyl; and C1-6alkoxycarbonyloxyethyl esters for example 1-methoxycarbonyloxyethyl and may be formed at any carboxy group in the compounds of this invention.
An in vivo hydrolysable ester of a compound of formula (A) or formula (I) containing a hydroxy group includes inorganic esters such as phosphate esters and xcex1-acyloxyalkyl ethers and related compounds which as a result of the in vivo hydrolysis of the ester breakdown to give the parent hydroxy group. Examples of xcex1-acyloxyalkyl ethers include acetoxymethoxy and 2,2-dimethylpropionyloxymethoxy. A selection of in vivo hydrolysable ester forming groups for hydroxy include alkanoyl, benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl, alkoxycarbonyl (to give alkyl carbonate esters), dialkylcarbamoyl and N-(dialkylaminoethyl)-N-alkylcarbamoyl (to give carbamates), dialkylaminoacetyl and carboxyacetyl.
Another aspect of the present invention provides a process for preparing a compound of formula (A) or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof which process comprises of:
a) reacting compounds of formula (IV): 
xe2x80x83where E is carboxy protected in the form of an ester and other groups are as defined in formula (A) with a compound of formula (V): 
xe2x80x83where L is a leaving group and other groups are as defined in formula (A) to give a compound of formula (VI): 
xe2x80x83where E is carboxy protected as an ester.
b) optionally interconverting a compound of formula (VI) to give another compound of formula (VI), wherein any functional groups are protected if necessary and optionally:
i) removing any protecting groups;
ii) optionally forming a pharmaceutically acceptable salt or in vivo hydrolysable ester.
Compounds of formula (VI) may be interconverted for example as described herein or by known processes such as functional group modification or aromatic substitution.
Preferred values for L are chloro and bromo. Preferred values for E are xe2x80x94CO2Et and xe2x80x94CO2Me.
Compounds of formula (IV) and (V) may be reacted together in an inert solvent and a base such as N,N-dimethylformamide/sodium hydride or dichloromethane/sodium hydroxide (optionally in the presence of a phase transfer catalyst such as tetra-n-butylammonium hydrogensulphate) for 1-6 hours preferably 1-3 hours, at a temperature of 15-30xc2x0 C., preferably 20-25xc2x0 C. to give a compound of formula (VI).
Compounds of formula (IV) are commercially available, made by modification using known processes of commercially available compounds of formula (IV), or they are prepared by:
a) Reacting a compound of formula (VII): 
xe2x80x83where R1 and p are as defined in formula (A), with a compound of formula (VIII) 
xe2x80x83where R8 is C1-4alkyl.
Compounds of formula (VII) and (VIII) are reacted together under Reissert reaction conditions such as in an inert solvent (such as tetrahydrofuran), in the presence of a base (such as potassium ethoxide), at a temperature range of 15-30xc2x0 C. preferably 20-25xc2x0 C., for 10-20 hours preferably 15-17 hours. The resulting compound is isolated and dissolved in an alcohol such as ethanol and an organic acid (such as acetic acid) and a transition metal catalyst (such as 10% Pd/C) and cyclohexene is added. The mixture is heated at a temperature of 60-120xc2x0 C. preferably at 70-90xc2x0 C. for 15-25 hours preferably 16-20 hours to give a compound of formula (IV); or
b) Reacting a compound of formula (IX): 
xe2x80x83where R1 and p are as defined for formula (A), with a compound of formula (X): 
xe2x80x83where R9 is C1-4alkyl.
Compounds of formula (IX) and (X) are reacted together under Fischer conditions such as with an organic acid (such as acetic acid), in an alcohol (such as ethanol), at a temperature of 60-90xc2x0 C., preferably 75-85xc2x0 C., for 1-5 hours, preferably 1-3 hours. The resulting compound is mixed with a strong acid (such as polyphosphoric acid) and heated at 90-150xc2x0 C. preferably 100-120xc2x0 C., for 0.5-4 hours, preferably 0.5-2 hours to give a compound of formula (IV) in which W is hydrogen. Then, if desired, W can be optionally converted into another value of W as defined in formula (A) using techniques known in the art such as those described below.
Compounds of formula (V), (VII), (VIII), (IX) and (X) are known or commercially available or are prepared by processes known in the art by standard manipulation of commercially available or known materials.
R8 and R9 are C1-4alkyl. Preferably R8 and R9 are methyl or ethyl.
It will be appreciated that analogous procedures to those described above may be used to prepare compounds of the formula (I), (Ixe2x80x2) and (B).
It will also be appreciated that certain of the various optional substituents in the compounds of formula (A), (B), (I) and (Ixe2x80x2) may be introduced by standard aromatic substitution reactions or generated by conventional functional group modifications either prior to or immediately following the processes mentioned above, and as such are included in the process aspect of the invention. Such reactions and modifications include, for example, introduction of a substituent by means of an aromatic substitution reaction, reduction of substituents, alkylation of substituents and oxidation of substituents. The reagents and reaction conditions for such procedures are well known in the chemical art. Particular examples of aromatic substitution reactions include the introduction of a nitro group using concentrated nitric acid, the introduction of an acyl group using, for example, an acylhalide and Lewis acid (such as aluminium trichloride) under Friedel Crafts conditions; the introduction of an alkyl group using an alkyl halide and Lewis acid (such as aluminium trichloride) under Friedel Crafts conditions; and the introduction of a halogeno group. Particular examples of modifications include the reduction of a nitro group to an amino group by for example, catalytic hydrogenation with a nickel catalyst or treatment with iron in the presence of hydrochloric acid with beating; oxidation of alkylthio to alkylsulphinyl or alkylsulphonyl. Specific examples of the substitution and modification reactions prior to or immediately following the processes mentioned above are illustrated, but not limited by, the following examples in which variable groups are as defined for formula (A) unless otherwise stated.
1) Modification of Ra.
a) For Ra=Ar (phenyl or thienyl): compounds of formula (XI) 
xe2x80x83where E is carboxy protected as an ester and M is H, a nitrogen protecting group or the group 
xe2x80x83and Ra is Br are coupled with compounds of formula (XII) 
xe2x80x83to give compounds of formula (XI) where Ra=Ar. It will be appreciated that an analogous procedure may be used to prepare compounds of formula (I) in which R1 is optionally substituted phenyl or optionally substituted 5 or 6 membered heteroaryl ring. Suitable reaction conditions are set out below.
Compounds of formula (XI) where Ra=Br and (XII) are reacted together in the presence of a transition metal catalyst (for example tetrakis(triphenylphosphine)palladium(0)), in an inert solvent (such as toluene) and an alcohol (such as ethanol), with an aqueous base (such as potassium carbonate), preferably in an inert atmosphere, at a temperature of 60-100xc2x0 C. preferably 75-85xc2x0 C. for 14-20 hours preferably 15-17 hours.
b) For Ra=NH2; compounds of formula (XI) where Ra=NO2 are reduced under standard conditions to give a compound of formula (XI) where Ra=NH2. Suitable reaction conditions are set out below.
Compounds of formula (XI) where Ra=NO2 are reacted with a reducing agent (such as sodium borohydride) and stannous chloride dihydrate in an alcohol (such as ethanol) at a temperature of 30-80xc2x0 C. preferably 50-70xc2x0 C. for 2-10 hours preferably 4-6 hours.
2) Hydrolysing a compound of formula (VI) as defined above to give a compound of formula (XIII): 
Suitable reaction conditions are set out below.
i) The general case where Xa is carboxy protected as an esterxe2x80x94in an inert solvent (such as tetrahydrofuran) and an alcohol (such as methanol), in the presence of a base (for example sodium hydroxide), at a temperature range of 10-50xc2x0 C. preferably 20-30xc2x0 C. for 1-25 hours preferably 15-20 hours followed by the addition of water and an acid (such as acetic acid).
ii) Specifically where Xa is xe2x80x94CO2Mexe2x80x94 with a salt (such as lithium iodide), in an organic base (such as pyridine), at a temperature range of 100-125xc2x0 C. especially 115-120xc2x0 C. for 3-10 hours preferably 5-7 hours followed by the addition of aqueous acid (for example 2M hydrochloric acid).
3) Modification of W.
a) For W=Br: compounds of formula (XI) where W=hydrogen may be brominated under standard conditions to give a compound of formula (XI) where W=Br. Suitable reaction conditions are set out below.
Compounds of formula (XI) where W=bromine may be prepared by reacting a compound of formula (XI) where W=hydrogen in an inert solvent (such as N,N-dimethylformamide) with bromine for 5-55 minutes particularly 25-35 minutes at 10-30xc2x0 C., preferably 20-25 xc2x0 C.
The reader is also directed to patent nos. US 005,389,650A, US 005,290,798A, EP 0535926A1, EP 0535923A1, US 005,190,968A, EP 0535924A1, EP 0419049A1, U.S. Pat. No. 5,308,850, EP 0535925A1, WO 93/16069, WO 93/25546, US 005,273,980A and U.S. Pat. No. 5,272,145, WO 93/20078, EP 0186367, EP 0275667, US 4,965,369A (process patent) WO 94/14434, EP 0480659 A2 and WO 96/03377A for synthetic details of benzyl indole compounds.
It will also be appreciated that in some of the reactions mentioned herein it may be necessary/desirable to protect any sensitive groups in the compounds. The instances where protection is necessary or desirable and suitable methods for protection are known to those skilled in the art. Thus, if reactants include groups such as amino, carboxy or hydroxy it may be desirable to protect the group in some of the reactions mentioned herein.
A suitable protecting group for an amino or alkylamino group is, for example, an acyl group, for example an alkanoyl group such as acetyl, an alkoxycarbonyl group, for example a methoxycarbonyl, ethoxycarbonyl or t-butoxycarbonyl group, an arylmethoxycarbonyl group, for example benzyloxycarbonyl, or an aroyl group, for example benzoyl. The deprotection conditions for the above protecting groups necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyl or alkoxycarbonyl group or an aroyl group may be removed for example, by hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium or sodium hydroxide. Alternatively an acyl group such as a t-butoxycarbonyl group may be removed, for example, by treatment with a suitable acid as hydrochloric, sulphuric or phosphoric acid or trifluoroacetic acid and an arylmethoxycarbonyl group such as a benzyloxycarbonyl group may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon, or by treatment with a Lewis acid for example boron tris(trifluoroacetate). A suitable alternative protecting group for a primary amino group is, for example, a phthaloyl group which may be removed by treatment with an alkylamine, for example dimethylaminopropylamine, or with hydrazine.
A suitable protecting group for a hydroxy group is, for example, an acyl group, for example an alkanoyl group such as acetyl, an aroyl group, for example benzoyl, or an arylmethyl group, for example benzyl. The deprotection conditions for the above protecting groups will necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyl or an aroyl group may be removed, for example, by hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium or sodium hydroxide. Alternatively an arylmethyl group such as a benzyl group may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon.
A suitable protecting group for a carboxy group is, for example, an esterifying group, for example a methyl or an ethyl group which may be removed, for example, by hydrolysis with a base such as sodium hydroxide, or for example a t-butyl group which may be removed, for example, by treatment with an acid, for example an organic acid such as trifluoroacetic acid, or for example a benzyl group which may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon.
The protecting groups may be removed at any convenient stage in the synthesis using conventional techniques well known in the chemical art.
When a pharmaceutically-acceptable salt of a compound of formula (A), formula (B) or formula (I) is required, it may be obtained, for example, by reaction of said compound with the appropriate acid (which affords a physiologically acceptable anion), or with the appropriate base (which affords a physiologically acceptable cation), or by any other conventional salt formation procedure.
When an optically active form of a compound of formula (I) is required, it may be obtained, for example, by carrying out one of the aforesaid procedures using an optically active starting material or by resolution of a racemic form of said compound using a conventional procedure.
According to a further aspect of the present invention there is provided a method for antagonising an MCP-1 mediated effect in a warm blooded animal, such as man, in need of such treatment, which comprises administering to said animal an effective amount of a compound of formula (A), formula (B) or formula (I), or a pharmaceutically acceptable salt, or an in vivo hydrolysable ester thereof.
According to a further feature of the invention there is provided a method of treatment of diseases or medical conditions mediated by MCP-1 which comprises administering to a warm-blooded animal an effective amount of a compound of formula (A), formula (B) or formula (I), or a pharmaceutically acceptable salt, or an in vivo hydrolysable ester thereof. According to a further aspect of the invention there is provided the use of a compound of the formula (I), (A) or (B) in the manufacture of a medicament for use in the treatment of a disease or medical condition mediated by MCP-1. Such diseases may include, for example, any of those previously referred to herein. According to a further aspect of the invention there is provided a compound of the formula (A) or (B), or a pharmaceutically acceptable salt thereof or an in vivo hydrolysable ester thereof, for use in a method of treatment of the human or animal body by therapy. According to a further aspect of the invention there is provided a method of inhibiting the binding of MCP-1 to a receptor thereof in a warm-blooded animal in need thereof which comprises administering to said warm-blooded animal an effective amount of a compound of formula (A), formula (B) or formula (I), or a pharmaceutically acceptable salt, or an in vivo hydrolysable ester thereof. According to a further aspect of the invention there is provided the use of a compound of the formula (I), (A) or (B) in the manufacture of a medicament for use in inhibiting the binding of MCP-1 to a receptor thereof.
In order to use a compound of formula (A), formula (B) or formula (I) or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof for the therapeutic treatment of mammals including humans, especially in treating inflammation, it is normally formulated in accordance with standard pharmaceutical practice as a pharmaceutical composition.
Therefore in another aspect the present invention provides a pharmaceutical composition which comprises a compound of formula (A) or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof and a pharmaceutically acceptable diluent or carrier. In another aspect the present invention provides a pharmaceutical composition which comprises a compound of formula (B) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable diluent or carrier.
The compositions of the invention may be in a form suitable for oral use (for example as tablets, lozenges, hard or soft capsules, aqueous or oily suspensions, emulsions, dispersible powders or granules, syrups or elixirs), for topical use (for example as creams, ointments, gels, or aqueous or oily solutions or suspensions), for administration by inhalation (for example as a finely divided powder or a liquid aerosol), for administration by insufflation (for example as a finely divided powder) or for parenteral administration (for example as a sterile aqueous or oily solution for intravenous, subcutaneous, intramuscular or intramuscular dosing or as a suppository for rectal dosing).
The compositions of the invention may be obtained by conventional procedures using conventional pharmaceutical excipients, well known in the art. Thus, compositions intended for oral use may contain, for example, one or more colouring, sweetening, flavouring and/or preservative agents.
Suitable pharmaceutically acceptable excipients for a tablet formulation include, for example, inert diluents such as lactose, sodium carbonate, calcium phosphate or calcium carbonate, granulating and disintegrating agents such as corn starch or algenic acid; binding agents such as starch; lubricating agents such as magnesium stearate, stearic acid or talc; preservative agents such as ethyl or propyl p-hydroxybenzoate, and anti-oxidants, such as ascorbic acid. Tablet formulations may be uncoated or coated either to modify their disintegration and the subsequent absorption of the active ingredient within the gastrointestinal track, or to improve their stability and/or appearance, in either case, using conventional coating agents and procedures well known in the art.
Compositions for-oral use may be in the form of hard gelatin capsules in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules in which the active ingredient is mixed with water or an oil such as peanut oil, liquid paraffin, or olive oil.
Aqueous suspensions generally contain the active ingredient in finely powdered form together with one or more suspending agents, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents such as lecithin or condensation products of an alkylene oxide with fatty acids (for example polyoxethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives (such as ethyl or propyl p-hydroxybenzoate, anti-oxidants (such as ascorbic acid), colouring agents, flavouring agents, and/or sweetening agents (such as sucrose, saccharine or aspartame).
Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil (such as arachis oil, olive oil, sesame oil or coconut oil) or in a mineral oil (such as liquid paraffin). The oily suspensions may also contain a thickening agent such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set out above, and flavouring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an anti-oxidant such as ascorbic acid.
Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water generally contain the active ingredient together with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients such as sweetening, flavouring and colouring agents, may also be present.
The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, such as olive oil or arachis oil, or a mineral oil, such as for example liquid paraffin or a mixture of any of these. Suitable emulsifying agents may be, for example, naturally-occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soya bean, lecithin, an esters or partial esters derived from fatty acids and hexitol anhydrides (for example sorbitan monooleate) and condensation products of the said partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening, flavouring and preservative agents.
Syrups and elixirs may be formulated with sweetening agents such as glycerol, propylene glycol, sorbitol, aspartame or sucrose, and may also contain a demulcent, preservative, flavouring and/or colouring agent.
The pharmaceutical compositions may also be in the form of a sterile injectable aqueous or oily suspension, which may be formulated according to known procedures using one or more of the appropriate dispersing or wetting agents and suspending agents, which have been mentioned above. A sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example a solution in 1,3-butanediol.
Suppository formulations may be prepared by mixing the active ingredient with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Suitable excipients include, for example, cocoa butter and polyethylene glycols.
Topical formulations, such as creams, ointments, gels and aqueous or oily solutions or suspensions, may generally be obtained by formulating an active ingredient with a conventional, topically acceptable, vehicle or diluent using conventional procedure well known in the art.
Compositions for administration by insufflation may be in the form of a finely divided powder containing particles of average diameter of, for example, 30xcexc or much less, the powder itself comprising either active ingredient alone or diluted with one or more physiologically acceptable carriers such as lactose. The powder for insulation is then conveniently retained in a capsule containing, for example, 1 to 50 mg of active ingredient for use with a turbo-inhaler device, such as is used for insufflation of the known agent sodium cromoglycate.
Compositions for administration by inhalation may be in the form of a conventional pressurised aerosol arranged to dispense the active ingredient either as an aerosol containing finely divided solid or liquid droplets. Conventional aerosol propellants such as volatile fluorinated hydrocarbons or hydrocarbons may be used and the aerosol device is conveniently arranged to dispense a metered quantity of active ingredient.
For further information on Formulation the reader is referred to Chapter 25.2 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.
The amount of active ingredient that is combined with one or more excipients to produce a single dosage form will necessarily vary depending upon the host treated and the particular route of administration. For example, a formulation intended for oral administration to humans will generally contain, for example, from 0.5 mg to 2 g of active agent compounded with an appropriate and convenient amount of excipients which may vary from about 5 to about 98 percent by weight of the total composition. Dosage unit forms will generally contain about 1 mg to about 500 mg of an active ingredient. For further information on Routes of Administration and Dosage Regimes the reader is referred to Chapter 25.3 in Volume 5 of Comprehensive Medicinal Chemistry (Corwin Hansch; Chairman of Editorial Board), Pergamon Press 1990.
The size of the dose for therapeutic or prophylactic purposes of a compound of the Formula I will naturally vary according to the nature and severity of the conditions, the age and sex of the animal or patient and the route of administration, according to well known principles of medicine. As mentioned above, compounds of the Formula I are useful in treating diseases or medical conditions which are due alone or in part to the effects of farnesylation of rats.
In using a compound of the Formula I for therapeutic or prophylactic purposes it will generally be administered so that a daily dose in the range, for example, 0.5 mg to 75 mg per kg body weight is received, given if required in divided doses. In general lower doses will be administered when a parenteral route is employed. Thus, for example, for intravenous administration, a dose in the range, for example, 0.5 mg to 30 mg per kg body weight will generally be used. Similarly, for administration by inhalation, a dose in the range, for example, 0.5 mg to 25 mg per kg body weight will be used. Oral administration is however preferred.
The following illustrate, but are not intended to limit, representative pharmaceutical dosage forms of the invention as defined herein (the active ingredient being termed xe2x80x9cCompound Xxe2x80x9d), for therapeutic or prophylactic use in humans:
Note
The above formulations may be obtained by conventional procedures well known in the pharmaceutical art. The tablets (a)-(c) may be enteric coated by conventional means, for example to provide a coating of cellulose acetate phthalate. The aerosol formulations (h)-(k) may be used in conjunction with standard, metered dose aerosol dispensers, and the suspending agents sorbitan trioleate and soya lecithin may be replaced by an alternative suspending agent such as sorbitan monooleate, sorbitan sesquioleate, polysorbate 80, polyglycerol oleate or oleic acid.
Biological Testing
The following biological test methods, data and Examples serve to illustrate the present invention.
Abbreviations:
AMPLITAQ(trademark), available from Perkin-Elmer Cetus, is used as the source of thermostable DNA polymerase.
Binding Buffer is 50 mM HEPES, 1 mM CaCl2, 5 mM MgCl2, 0.5% foetal calf serum, adjusted to pH 7.2 with 1 M NaOH.
Non-Essential Amino Acids (100xc3x97concentrate) is: L-Alanine, 890 mg/l; L-Asparagine, 1320 mg/l; L-Aspartic acid, 1330 mg/l; L-Glutamic acid, 1470 mg/l; Glycine, 750 mg/l; L-Proline, 1150 mg/l and; L-Serine, 1050 mg/l.
Hypoxanthine and Thymidine Supplement (50xc3x97concentrate) is: hypoxanthine, 680 mg/l and; thymidine, 194 mg/l.
Penicillin-Streptomycin is: Penicillin G (sodium salt); 5000 units/ml; Streptomycin sulphate, 5000 xcexcg/ml.
Human monocytic cell line THP-1 cells are available from ATCC, accession number ATCC TIB-202.
Hank""s Balanced Salt Solution (HBSS) was obtained from Gibco; see Proc. Soc. Exp. Biol. Med., 1949, 71, 196.
Synthetic cell culture medium, RPMI 1640 was obtained from Gibco; it contains inorganic salts [Ca(NO3)2.4H2O 100 mg/l; KCl 400 mg/l; MgSO4.7H2O 100 mg/l; NaCl 6000 mg/l; NaHCO3 2000 mg/l and Na2HPO4 (anhyd) 800 mg/l], D-Glucose 2000 mg/l, reduced glutathione 1 mg/l, amino acids and vitamins.
FURA-2/AM is
1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2xe2x80x2-amino-5xe2x80x2-methylphenoxy)-ethane-N,N,Nxe2x80x2,Nxe2x80x2-tetraacetic acid pentaacetoxymethyl ester and was obtained from Molecular Probes, Eugene, Oreg., USA.
General molecular biology procedures can be followed from any of the methods described in xe2x80x9cMolecular Cloningxe2x80x94A Laboratory Manualxe2x80x9d Second Edition, Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory, 1989).
Biological Assays for hMCP-1 Antagonists
a) hMCP-1 Receptor-binding assay
i) Cloning and expression of hMCP-1 receptor
The MCP-1 receptor B (CCR2B) cDNA was cloned by PCR from THP-1 cell RNA using suitable oligonucleotide primers based on the published MCP-1 receptor sequences (Charo et al., 1994, Proc. Natl. Acad. Sci. USA, 91, 2752). The resulting PCR products were cloned into vector PCR-II(trademark) (InVitrogen, San Diego, Calif.). Error free CCR2B cDNA was subcloned as a Hind III-Not I fragment into the eukaryotic expression vector pCDNA3 (InVitrogen) to generate pCDNA3/CC-CKR2A and pCDNA3/CCR2B respectively.
Linearised pCDNA3/CCR2B DNA was transfected into CHO-K1 cells by calcium phosphate precipitation (Wigler et al., 1979, Cell, 16, 777). Transfected cells were selected by the addition of Geneticin Sulphate (G418, Gibco BRL) at 1 mg/ml, 24 hours after the cells had been transfected. Preparation of RNA and Northern blotting were carried out as described previously (Needham et al., 1995, Prot. Express. Purific., 6, 134). CHO-K1 clone 7 (CHO-CCR2B) was identified as the highest MCP-1 receptor B expressor.
ii) Preparation of membrane fragments
CHO-CCR2B cells were grown in DMEM supplemented with 10% foetal calf serum, 2 mM glutamine, 1xc3x97 Non-Essential Amino Acids, 1xc3x97 Hypoxanthine and Thymidine Supplement and Penicillin-Streptomycin (at 50 xcexcg streptomycin/ml, Gibco BRL). Membrane fragments were prepared using cell lysis/differential centrifugation methods as described previously (Siciliano et al., 1990, J. Biol. Chem., 265, 19658). Protein concentration was estimated by BCA protein assay (Pierce, Rockford, Ill.) according to the manufacturer""s instructions.
iii) Assay
125I MCP-1 was prepared using Bolton and Hunter conjugation (Bolton et al., 1973, Biochem. J., 133, 529; Amersham International plc]. Equilibrium binding assays were carried out using the method of Ernst et al., 1994, J. Immunol., 152, 3541. Briefly, varying amounts of 125I-labeled MCP-1 were added to 10 mg of purified CHO-CCR2B cell membranes in 100 ml of Binding Buffer. After 1 hour incubation at room temperature the binding reaction mixtures were filtered and washed 5 times through a plate washer (Packard Harvester Filtermate(trademark) 196). Scintillation fluid (25 xcexcl, Microscint(trademark)-20, a high efficiency liquid scintillation counting cocktail for aqueous samples) was added to each well and the plate was covered with plate sealer and counted (Packard Top Count(trademark)). Cold competition studies were performed as above using 100 pM 125I-labeled MCP-1 in the presence of varying concentrations of unlabelled MCP-1. Non-specific binding was determined by the inclusion of a 200-fold molar excess of unlabelled MCP-1 in the reaction.
Ligand binding studies with membrane fragments prepared from CHO-CCR2B cells showed that the CCR2B was present at a concentration of 0.2 pmoles/mg of membrane protein and bound MCP-1 selectively and with high affinity (IC50=110 pM, Kd=120 pM). Binding to these membranes was completely reversible and reached equilibrium after 45 minutes at room temperature, and there was a linear relationship between MCP-1 binding and CHO-CCR2B cell membrane concentration when using MCP-1 at concentrations between 100 pM and 500 pM.
Test compounds dissolved in DMSO (5 xcexcl) were tested in competition with 100 pM labelled MCP-1 over a concentration range (0.1-200 xcexcM) in duplicate using eight point dose-response curves and IC50 concentrations were calculated.
b) MCP-1 mediated calcium flux in THP-1 cells
The human monocytic cell line THP-1 was grown in a synthetic cell culture medium RPMI 1640 supplemented with 10% foetal calf serum, 2 mM glutamine and Penicillin-Streptomycin (at 50 xcexcg streptomycin/ml, Gibco BRL). THP-1 cells were washed in HBSS (lacking Ca2+ and Mg2+)+1 mg/ml BSA and resuspended in the same buffer at a density of 3xc3x97106 cells/ml. The cells were then loaded with 1 mM FURA-2/AM for 30 min at 37xc2x0 C., washed twice in HBSS, and resuspended at 1xc3x97106 cells/ml. THP-1 cell suspension (0.9 ml) was added to a 5 ml disposable cuvette containing a magnetic stirrer bar and 2.1 ml of prewarmed (37xc2x0 C.) HBSS containing 1 mg/ml BSA, 1 mM MgCl2 and 2 mM CaCl2. The cuvette was placed in a fluorescence spectrophotometer (Perkin Elmer, Norwalk, Conn.) and preincubated for 4 min at 37xc2x0 C. with stirring. Fluorescence was recorded over 70 sec and cells were stimulated by addition of hMCP-1 to the cuvette after 10 sec. [Ca2+]i was measured by excitation at 340 nm and 380 nm alternately and subsequent measurement of the intensity of the fluorescence emission at 510 nm. The ratio of the intensities of the emitted fluorescent light following excitation at 340 nm and 380 nm, (R), was calculated and displayed to give and estimate of cytoplasmic [Ca2+] according to the equation:             [              Ca                  2          +                    ]        ⁢    i    =            K      d        ⁢                  (                  R          -                      R            ⁢                          xe2x80x83                        ⁢            min                          )                    (                              R            ⁢                          xe2x80x83                        ⁢            max                    -          R                )              ⁢          (              Sf2        /        Sb2            )      
where the Kd for FURA-2 Ca2+ complex at 37xc2x0 C. was taken to be 224 nm. Rmax is the maximal fluorescence ratio determined after addition of 10 mM Ionomycin, Rmin is the minimal ratio determined by the subsequent addition of a Ca2+ free solution containing 5 mM EGTA, and Sf2/Sb2 is the ratio of fluorescence values at 380 nm excitation determined at Rmin and Rmax, respectively.
Stimulation of THP-1 cells with HMCP-1 induced a rapid, transient rise in [Ca2+]i in a specific and dose dependent manner. Dose response curves indicated an approximate EC50 of 2 nm. Test compounds dissolved in DMSO (10 xcexcl) were assayed for inhibition of calcium release by adding them to the cell suspension 10 sec prior to ligand addition and measuring the reduction in the transient rise in [Ca2+]. Test compounds were also checked for lack of agonism by addition in place of hMCP-1.
c) hMCP-1 mediated chemotaxis assay
In vitro chemotaxis assays were performed using either the human monocytic cell line THP-1 or peripheral blood mixed monocytes obtained from fresh human blood purified by erythrocyte sedimentation followed by density gradient centrifugation over 9.6% (w/v) sodium metrizoate and 5.6% (w/v) polysaccharide, density 1.077 g/ml (Lymphopre(trademark) Nycomed). Cell migration through polycarbonate membranes was measured by enumerating those passing through either directly by Coulter counting or indirectly by use of a colourimetric viability assay measuring the cleavage of a tetrazolium salt by the mitochondrial respiratory chain (Scudiero D. A. et al. 1988, Cancer Res., 48, 4827-4833).
Chemoattractants were introduced into a 96-well microtiter plate which forms the lower well of a chemotaxis chamber fitted with a PVP-free 5 xcexcm poresize polycarbonate adhesive framed filter membrane (NeuroProbe MB series, Cabin John, Md. 20818, USA) according to the manufacturer""s instructions. The chemoattractant was diluted as appropriate in synthetic cell culture medium, RPMI 1640 (Gibco) supplemented with 2 mM glutamine and 0.5% BSA. Each dilution was degassed under vacuum for 30 min and was placed (400 xcexcl) in the lower wells of the chamber and THP-1 cells (5xc3x97105 in 100 xcexcl RPMI 1640+0.5% BSA) were incubated in each well of the upper chamber. For the inhibition of chemotaxis the chemoattractant was kept at a constant submaximal concentration determined previously for each chemokine and added to the lower well together with the test compounds dissolved in DMSO (final DMSO concentration  less than 0.05% v/v) at varying concentrations. The chamber was incubated for 2 h at 37xc2x0 C. under 5% CO2. The medium was removed from the upper wells which were then washed out with 200 xcexcl physiological saline before opening the chamber, wiping dry the membrane surface and centrifuging the 96-well plate at 600 g for 5 min to harvest the cells. Supernatant (150 xcexcl) was aspirated and 10 xcexcl of cell proliferation reagent, WST-1, {4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate} plus an electron coupling reagent (Boehringer Mannheim, Cat.no. 1644 807) was added back to the wells. The plate was incubated at 37xc2x0 C. for 3 h and the absorbance of the soluble formazan product was read on a microtitre plate reader at 450 nm. The data was input into a spreadsheet, corrected for any random migration in the absence of chemoattractant and the average absorbance values, standard error of the mean, and significance tests were calculated. hMCP-1 induced concentration dependent cell migration with a characteristic biphasic response, maximal 0.5-1.0 nm.
Compounds tested of the present invention generally had IC50 values of less than 50 xcexcM in the hMCP-1 receptor binding assay described herein. For example the compound of example 3.23 had an IC50 of 7.38 xcexcM.